Transient Disruption of Intercellular Junctions Enables Baculovirus Entry into Nondividing Hepatocytes

Abstract

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…In either medium condition, a dose-dependent increase in the percentage of cells expressing b-gal was observed. As can be seen from Figure 1 and was previously reported, 14 in the presence of calcium, baculovirus-mediated gene transfer to long-term cultures of primary rat hepatocytes was limited to hepatocytes on the periphery of the islands; at an moi of 1600 PFU/cell, the percentage of hepatocytes expressing the delivered b-gal transgene was approximately 10%. 14 However, in cultures pretreated and infected with 1600 PFU CMV-lacZ baculovirus/cell in Figure 1 Depletion of extracellular calcium conferred increased baculovirus-mediated gene delivery to long-term primary rat hepatocytes.…”

“…As can be seen from Figure 1 and was previously reported, 14 in the presence of calcium, baculovirus-mediated gene transfer to long-term cultures of primary rat hepatocytes was limited to hepatocytes on the periphery of the islands; at an moi of 1600 PFU/cell, the percentage of hepatocytes expressing the delivered b-gal transgene was approximately 10%. 14 However, in cultures pretreated and infected with 1600 PFU CMV-lacZ baculovirus/cell in Figure 1 Depletion of extracellular calcium conferred increased baculovirus-mediated gene delivery to long-term primary rat hepatocytes. At 20 days postseeding, primary rat hepatocytes in long-term DMSO culture were pretreated in either control medium or calcium-free DMEM supplemented with 100 mM EGTA for 1 h. Subsequently, cultures were either mock-infected or infected for 1 h with 400, 800, or 1600 PFU CMVlacZ baculovirus/cell diluted in either control medium or calcium-free DMEM supplemented with 100 mM EGTA.…”

“…2,5 The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can efficiently deliver genes to numerous mammalian cell types including hepatic cells. [6][7][8][9][10][11][12][13][14] Recently, we have characterized baculovirusmediated gene delivery into long-term primary rat hepatocytes maintained in a chemically defined medium supplemented with DMSO. 14 Baculovirus-mediated gene transfer in this culture system was dose-dependent and restricted to peripheral cells of hepatocellular islands when extracellular calcium was present during baculovirus infection.…”

“…24 Several studies have determined that disruption of calcium-dependent paracellular junction complexes by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 14,23,[25][26][27][28] Transient depletion of extracellular calcium may overcome the inefficiency of baculovirus-mediated gene transfer by disassociating paracellular epithelial junction complexes and exposing the basolateral surface. We hypothesized that in normal medium, which contains calcium, the baculovirus binds and delivers its genome only to peripheral cells and baculovirus attachment, and entry to internal cells is prevented because paracellular junctions mask the baculovirus binding motif of these internal hepatocytes.…”

“…14 However, to measure the value of this novel technology for gene transfer for in vitro and translative in vivo purposes, it is important to determine (i) the effects of calcium depletion on hepatocyte paracellular junctions that enable the baculovirus to enter the hepatocytes, (ii) the time course and reversible nature of these processes, and (iii) the effect of this gene transfer method on two liver-specific characteristics of hepatocytes, ultrastructure, and albumin gene expression. These goals are addressed in this study.…”